A Study On Genetic Transformation Of Vitreoscilla Hemoglobin Gene (VHb) In Maize (Zea Mays L.)

Waterlongging is one of the serious abiotic stresses which lead to loss of grain yield and limit the area of crop growing. Breeding of waterlogging toerance varieties is a available strategy for decreasing loss of yield of crop. It is known that the VHb gene encoding a Vitreoscilla hemoglobin can is expressed in plant under oxygen limited condition and then effectively improves waterlogging tolerance of plant. Thus, transfer of VHb gene into maize by particle bombardment and breeding of transgenic inbred of maize is significant to develop new germplasm of maize and improvement of waterlogging tolerance of maize. the A188 x B73 were used as the material,the callus as the receptor, VHb gene were transduced to receptor cell by bombardment transformation.The major results are as follows:1. The induction of transgenic seedling foot system were affected by many tissue culture fctors.The impact of different types of medium on survival rate, root length, leaf length,number of leaf,seeding height is significantly different.In the five medium used,1/2MS(Murashige and Skoog medium)+0.3NAA had the highest survival rate,which reached 75 percent.The survival rate of 1/2MS was lowest and was merely 33.3 percent.In the process of root regenerating,1/2MS medium supplemented with 0.3mg/L of NAA can promote the regeneration of root, and increase the number of roots, so it can be regard as a more suitable rooting medium.2. In this study, with bar gene as the selection marker, 31 resistant calli were obtained after 3 rounds of screening, in which 29 resistant calli appeared blue in GUS activity detection. 94 strains were obtained in T0 generation transgenic plants, of which 54 strains were PCR positive, account for 57.4 percent of resistant plant,and ultimately five plants had ears.Through further RT–PCR of maize leaves from positive plants, 7 plants of the 10 transgenic regenerated plants were positive, the result showed that VHb gene was integrated into the maize genome, and some transgenic plants had the expression of transcriptional level.3. A large amount of sterile plants appeared in every transgenic generation by field observation and the I–KI pollen staining.There is no significant difference between negative plants and positive plants in the same incident. PCR–positive plants also had both fertile plants and sterile plants, the negative plants alike. These results certified that sterile variation of regeneration plant was arose by tissue culture, and belonged to somaclonal variation. 4. The PCR detection of T1 transgenic plants show that 11 plants were positive,26 plants were negative.The positive rate was 42.3 percent,which suggested that VHB gene had been stablely intergrated into the genome of maize,and expressed in transgenic plants.